This invention relates to collagen constructs and to methods of making and using such constructs. This invention further relates to tissue equivalents having improved characteristics and to methods of making and using such tissue equivalents. This invention also relates to methods of producing highly concentrated solutions of collagen.
Collagen is usually found as the principal protein component of the extra-cellular matrix. In mammals, collagen sometimes constitutes as much as 60% of the total body protein. It comprises most of the organic matter of skin, tendons, bones and teeth, and occurs as fibrous inclusions in most other body structures. Collagen is a relatively weak immunogen, due in part to masking of potential antigenic determinants by the helical structure. This helical structure also makes collagen resistant to proteolysis. Collagen is a natural substance for cell adhesion and the major tensile load-bearing component of the musculo-skeletal system.
Because of the foregoing properties, collagen has applications in the manufacture of implantable prostheses, as a cell growth substrate, and in the preparation of living tissue equivalents. Much work has been done to develop collagen constructs for such applications, including constructs for use in research and development, tissue and organ repair and/or replacement. Collagen is the principal protein component of such collagen constructs.
Many methods are known for organizing collagen into constructs such as injectable pastes, living tissue equivalents, films, sponges and so forth. These methods include the formation of collagen fibrils for injectable pastes and blood vessel prosteheses, e.g., U.S. Pat. Nos. 4,252,759; 4,787,900; 4,319,363; and 3,425,418; the formation of collagen films, e.g., U.S. Pat. No. 3,014,024; and the formation of sponges, e.g., U.S. Pat. No. 4,320,201.
Another method of forming collagen constructs involves the contraction of collagen gels by a contractile agent, such as fibroblast cells, smooth muscle cells or blood platelets, to form living tissue equivalents.. Such tissue equivalents are disclosed in U.S. Pat. Nos. 4,485,096; 4,485,097; 4,539,716; 4,546,500; 4,604,346; 4,835,102; and 4,837,379 and co-pending application U.S. Ser. No. 07/252,249, filed Sep. 30, 1988, all of which are incorporated herein by reference (hereinafter collectively referred to as "the Patents"). These tissue equivalents include, but are not limited to, equivalents of epithelial tissue and connective tissue such as skin, cartilage, bone, blood vessels, and comprise living cells and extracellular matrix molecules, principally collagen, and may optionally be provided with components not typically found in normal tissue. Such tissue equivalents have a broad range of applications including applications in research and development, tissue and organ replacement and testing.
In these known methods, the collagen construct is organized from relatively dilute solutions of collagen, e.g., about 5-10 mg/ml, by air-drying or neutralizing. The collagen constructs which are produced by such methods typically have a sparse collagen density and few collagen/collagen interactions. These characteristics tend to decrease the structural integrity of such constructs. Moreover, many known methods of preparing collagen constructs also suffer the disadvantage of a lack of flexibility and in the degree of control over the process to form a construct having the desired shape.
It is highly desirable that the strength of such constructs be sufficient to enable ease of handling and to provide durability, particularly in applications which involve a significant mechanical handling or tensile or pulsatile stress. Accordingly, it is desirable to form collagen constructs which have a more dense fibrillar structure more akin to those found in vivo. It is believed that in vivo collagen is organized from very concentrated solutions. Thus, improved collagen constructs and methods of preparing such constructs are being sought.